Western blot protocol- Cell Lysis Buffers

(Solutions and reagents to go with your Western blot protocol)

Choosing the correct Cell Lysis Buffer is a critical step in developing a successful Western blot protocol.  Often this will require you to try several lysis buffers to find the best one for releasing your protein/proteins of choice so they can be run a SDS-Page gel, transfered to a membrane, and ultimately recognized by a primary antibody.

Most antibodies will recognize reduced and denatured proteins and meaning your protein should be kept under reducing and denaturing conditions (Duh). Occasionally, antibodies will only recognize a protein in its native, non-denatured form forcing you to resort to non-reducing conditions. Take careful note of these terms before you buy a Western blot antibody (check the antibody datasheet for words like mild detergent or without detergent, or run a quick full text search with your antibody in our full text search engine).

Cells Lysis buffers for Western blotting

We typically store these buffers at 4°C for several weeks. But don’t get lazy! It makes no sense to waste a week troubleshooting Western blots because you were too lazy spend 20 min making fresh lysis buffers.

1X SDS Sample Buffer – “Like a hammer -good for pounding”
62.5 mM Tris-HCl (pH 6.8 at 25°C)
2% w/v SDS, 10% glycerol,
50 mM DTT
0.01% w/v bromophenol blue

RIPA buffer – good general choice for Whole Cell Lysates, Nuclear, mitochondrial, or membrane proteins
150 mM NaCl
1.0% NP-40 or 0.1% Triton X-100
0.5% sodium deoxycholate
0.1% SDS (sodium dodecyl sulphate)
50 mM Tris-HCl pH 8.0
add fresh Protease Inhibitors

**Note RIPA buffer will disrupt protein-protein interactions, so no immunoprecipitation reactions or pull down assays.

NP40 buffer – good for antibodies that react to non-denatured proteins
150 mM NaCl
1.0% NP-40 (Nonidet-P40)
50 mM Tris-HCl pH 8.0
add fresh Protease Inhibitors

NP-40 is a good general buffer for studying proteins that are easily released from cells, cytoplasm, or other proteins. It will not disrupt protein-protein interactions.

Tris-Triton buffer – Proteins bound to cytoskeleton in cytoplasm
10 mM Tris, pH 7.4
100 mM NaCl
1 mM EDTA
1 mM EGTA
1% Triton X-100
10% glycerol
0.1% SDS
0.5% deoxycholate
add fresh Protease Inhibitors

Tris-HCl buffer – Soluble cytoplasmic proteins only
20 mM Tris-HCl pH 7.5
add fresh Protease Inhibitors